N. M. Crawford, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA Fax/Tel: +1 858 534 1637 E-mail: email@example.com
There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of l-citrulline from l-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 µm), implicating the urea cycle enzyme argininosuccinate lyase (EC 188.8.131.52), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.
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Nitric oxide (NO) serves as a central signal in a wide variety of processes, including vasodilation, neural communication and immune function in animals , and defense responses, hormonal signaling and flowering in plants [2–6]. The primary mechanism for NO synthesis in animals involves oxidation of l-arginine to l-citrulline and NO, and requires NADPH and oxygen [7–9]. This reaction is catalyzed by nitric oxide synthase (NOS) enzymes, which require tetrahydrobiopterin (BH4), FMN, FAD, calmodulin (CaM), and Ca2+. Three isoforms of highly conserved NOS enzymes have been identified in mammals: neuronal NOS (nNOS or NOS-I), inducible NOS (iNOS or NOS-II), and endothelial NOS (eNOS or NOS-III). NOS enzymes contain an N-terminal oxygenase domain and a C-terminal reductase domain connected by a CaM-binding hinge region. NOS enzymes are also found in specific species of fish, invertebrates, protozoa and fungi [10–12]. Even bacteria contain genes coding for truncated NOS proteins with homology to the oxygenase domain of mammalian NOS, and these enzymes have nitration or NO synthesis activity [13–16].
Despite the high degree of conservation found among NOS enzymes, no protein with significant sequence similarity has been identified in plants, including Arabidopsis and rice , the genomes of which have been sequenced. Plants can produce and release significant amounts of NO, especially under hypoxic conditions or during infection [2,3,19–27]. One source of NO is nitrite, which can be converted to NO by: (a) plant nitrate reductase [22,28–30]; (b) mitochondria [31–33]; and (c) nonenzymatic processes [34,35]. There is also ample evidence from biochemical and pharmacological data that an arginine-dependent mechanism analogous to animal NOS reactions exists in plants [26,36–43]; however, the identity of the arginine-dependent activity in plants has yet to be conclusively determined.
Some of the evidence supporting an arginine-dependent mechanism in plants comes from commercially available ‘NOS assay kits’ (citrulline-based assays) that measure the conversion of arginine to citrulline using ion exchange chromatography . Radiolabeled arginine is provided as a substrate, and is then separated from reaction products by cation exchange chromatography. Positively charged arginine binds the ion exchange resin but citrulline does not. The unbound fraction, which is generally assumed to be citrulline, is measured in a scintillation counter. Examples of the use of this assay include the analysis of NOS activity in aluminum-treated Hibiscus, in pea peroxisomes , and in elicitor-treated Hypericum cells . Although the assay is quick and sensitive, it does not identify the product as citrulline; any arginine derivative that does not bind to the cation exchange resin will give a signal. The discovery of a product from a typical NOS reaction that is not citrulline was reported in a mammalian system .
There have been several attempts to identify the source responsible for arginine-dependent NOS activity in plants. The most recent attempt, which identified the gene AtNOS1, has subsequently been challenged [48–50], leading to the proposal that the gene be renamed AtNOA1 for nitric oxide-associated . Thus, a renewed effort was made to determine the source of arginine-dependent NOS activity in plants, using crude protein extracts from Arabidopsis leaves. By employing the citrulline-based NOS assay, an arginine-dependent activity was discovered that was strongly stimulated by an extract of low molecular weight compounds from Arabidopsis leaves and produced argininosuccinate rather than citrulline. These results identify a reaction that is catalyzed by an activity unrelated to NOS and that can interfere with or mask authentic NOS activity.
Results and Discussion
As a first approach to search for NOS activity in Arabidopsis, the citrulline-based NOS assay was used to test extracts from Arabidopsis leaves. Crude protein extracts (supernatant from a 2 × 104g centrifugation) were incubated with [14C]arginine, NADPH and mammalian NOS cofactors (BH4, FMN, FAD, Ca2+ and CaM). At the end of the reaction, unreacted arginine was removed from the assay mixture with a cation exchange resin. Radioactive material that did not bind the resin, presumably citrulline, was measured by scintillation counting. The signal obtained from a complete reaction (Fig. 1, lane 1) was up to 20 times higher than that from the control, which was a complete reaction terminated immediately after the addition of radiolabeled arginine. To determine potential cofactor requirements for the observed activity, leaf extracts were desalted using G-25 Sephadex to remove low molecular weight compounds. The low molecular weight compounds retained by the G-25 column were also collected by further elution of the column as described in Experimental procedures. Desalted protein extracts alone had greatly reduced levels of activity (Fig. 1, lane 2), indicating that a low molecular weight compound(s) from the extract was necessary for activity. Adding back the low molecular weight fraction from the G-25 column to the desalted protein extract restored activity (Fig. 1, lane 3). We named the low molecular weight fraction ADF, for Arabidopsis-derived factor. The stimulation of activity by ADF was positively correlated with the amount of ADF added (Fig. 2). Boiled protein extract showed very little activity in the presence of ADF (Fig. 1, lane 4), whereas boiled ADF (Fig. 1, lane 5) stimulated activity as much as untreated ADF (Fig. 1, lane 3) when added to the desalted extract, indicating that ADF is heat stable.
These results suggested that an arginine-dependent activity was present in protein extracts of Arabidopsis leaves, and that a low molecular weight molecule(s) was required for this activity. To determine whether this activity was similar to that of mammalian NOS enzymes, two experiments were performed. First, cofactors essential for NOS activity (BH4, FMN, FAD, Ca2+ and CaM) were omitted from the reaction. Robust activity was still observed for crude protein extract and desalted protein extract to which ADF was added (Fig. 3A, lanes 1–4). For these reactions, partially purified ADF preparations were used (purification involved boiling leaf extracts and then passing them through two gel filtration columns and an anion exchange column, as described in Experimental procedures). We performed an additional experiment to test for flavin-dependent activity, using diphenylene iodonium (an inhibitor of flavoproteins including animal NOS), and found no inhibition of the activity at concentrations of diphenylene iodonium up to 10 µm (data not shown). Second, the products of the reaction were analyzed by one-dimensional TLC followed by autoradiography. No citrulline was detected on the autoradiograms; instead, an unidentified compound was observed as the major reaction product (Fig. 3B). Together, these results showed that the reaction had no requirement for known NOS cofactors and did not produce the NOS coproduct citrulline, indicating that it was not a typical NOS reaction.
To identify the unknown compound, the reaction products were analyzed by two-dimensional TLC on silica gel plates. 14C-Labeled argininosuccinate was the only radiolabeled product identified (Fig. 4). No radiolabeled products comigrating with citrulline, ornithine, urea, valine, hydroxyarginine, agmatine, spermine, spermidine, putrescine or proline were detected (Fig. 4 and data not shown).
Argininosuccinate is the immediate precursor to arginine in the urea cycle, and is converted to arginine and fumarate by argininosuccinate lyase (ASL; EC 184.108.40.206; Fig. 5). Argininosuccinate is normally made from citrulline and aspartate by argininosuccinate synthetase, but it can also be produced by ASL in a reverse reaction. ASL is found in plants, animals and bacteria, and requires no external cofactors or metal ions for catalytic activity . The forward reaction (argininosuccinate to arginine and fumarate) is favored; reported Km values for argininosuccinate range from 0.13 mm in jack bean  to 0.2 mm in human liver , whereas the reported Km values for the reverse reaction are 5.3 mm for fumarate and 3.0 mm for arginine .
If argininosuccinate synthesis is being catalyzed by ASL in the Arabidopsis protein extracts, then fumarate would be needed as a cosubstrate, and fumarate would be the active component in the ADF preparation. Therefore, partially purified ADF was treated with fumarase, which converts fumarate to malate. After fumarase treatment, ADF no longer enhanced the production of argininosuccinate (Table 1). Next, fumarate was tested as a replacement for ADF in the reactions. Desalted protein extracts from Arabidopsis were incubated with either ADF or fumarate; both reactions produced the same product, which comigrated with argininosuccinate by TLC analysis (Fig. 6). When maleic acid (the cis-isomer of fumarate) was used instead of fumarate, no activity was detected (data not shown). When the amount of product produced was measured as a function of fumarate concentration using desalted Arabidopsis extracts, the data showed a saturation curve (Fig. 7), which yielded a Km (fumarate) of 4.5 mm, similar to what is reported for human liver . The reaction could be strongly inhibited (by 97%) by 0.3 mm argininosuccinate (data not shown), the substrate for the favored forward reaction. Desalted protein extracts from Escherichia coli were also tested, and the same argininosuccinate product was produced with ADF or fumarate (Fig. 6).
Table 1. Fumarase destroys ADF activity. [14C]Arginine was incubated with desalted protein extracts from Arabidopsis leaves and partially purified ADF that was untreated or treated with fumarase as indicated. Treated ADF (500 µL) was incubated with 50 U of fumarase, and an aliquot was used in the assay after fumarase was inactivated by heat. Activity is presented as delta c.p.m.·mg−1 protein·h−1 with SDs.
Desalted extract + ADF
Desalted extract + treated ADF
16 354 ±1267
16 085 ±1440
Our results show that when the citrulline-based assay is employed, protein extracts from Arabidopsis catalyze a reaction with arginine that mimics an NOS reaction. This reaction, however, produces argininosuccinate, not citrulline, and requires fumarate, indicating that ASL is catalyzing the reaction. Because argininosuccinate does not bind the cation exchange column, the signal from the reaction could be mistaken for NOS activity. Initially, it was puzzling why activity was obtained in crude Arabidopsis extracts without added fumarate (ADF); however, several articles have reported that fumarate levels can be quite high in plants, especially in Arabidopsis, where it is reported to be one of the most abundant organic acids [54,55]. The same activity can also be observed in protein extracts of E. coli, but only if fumarate or low molecular weight compounds from Arabidopsis leaves are added to the E. coli extracts.
These results demonstrate the importance of verifying the identity of the products in standard citrulline-based NOS assays of plant and, especially, Arabidopsis extracts. Until such tests are performed, the results from such assays cannot be used to support the existence of arginine-dependent NOS activity in plants.
Plant material and protein extractions
Leaves from 3-week old Arabidopsis plants (ecotype Columbia) grown under 16 h light conditions were harvested and ground in liquid N2 with a mortar and pestle. Extraction buffer (2.5 mL of 50 mm Hepes, pH 7.4, 1 mm EDTA, 10 mm MgCl2, 1 mmβ-mercaptoethanol, 1 mm 4-(2-aminoethyl)-benzolsulfonylfluorid, 1 × Roche Protease Inhibitor cocktail per gram fresh weight) was mixed with the ground plant material, and samples were centrifuged (2 × 104g) for 10 min at 4 °C (Beckman J2-HS, rotor JA-20, Palo Alto, CA, USA). The supernatant (crude protein extract) was either used directly or further desalted on a G-25 Sephadex gel filtration column (PD-10 column from GE Healthcare, Piscataway, NJ, USA), according to the manufacturer's instructions. Briefly, 2.5 mL protein extract was applied to a PD10 column of 10 mL bed volume and then washed with extraction buffer. The first 2.5 mL of eluant was discarded, the next 3.5 mL (excluded volume) was collected (called desalted protein extract), and the next 3.5 mL (included volume) was collected and contained small molecules. Extracts were concentrated in a Centricon-30 filter device (Millipore, Bedford, MA, USA) at 4 °C. For E. coli protein extracts, cell pellets were resuspended in lysis buffer (25 mm Hepes, 0.7 mm Na2HPO4, 137 mm NaCl, 5 mm KCl, pH 7.4), incubated on ice for 20 min with 1 mg·mL−1 lysozyme, and sonicated. Lysate was centrifuged at 100 000 g for 1 h (Beckman ultracentrifuge L7, rotor SW51), desalted on a PD-10 column, and concentrated with a Centricon-30 filter device. Protein concentrations were determined using the Bradford Assay (Biorad, Hercules, CA, USA).
Leaf tissue (50 g) from 3-week-old Arabidopsis plants was boiled for 15 min in 100 mL of water containing 1 mmβ-mercaptoethanol. The boiled extract was centrifuged at 2 × 104g at room temperature (Beckman J2-HS, rotor JA-20), and the supernatant was lyophilized. Resuspended material was used directly or partially purified on a 72 cm × 1.5 cm column containing G-15 Sephadex (Sigma, St Louis, MO, USA) in water. Fractions were assayed for activation of desalted protein extracts. Active fractions were subsequently pooled and applied to a Q-Separose FF column (Amersham) equilibrated with 50 mm NaPO4 (pH 7.4). The column was eluted with increasing concentrations of NaCl. Active fractions eluted between 0.4 m and 0.5 m NaCl. These fractions were pooled, lyophilized, and separated on the same G-15 Sephadex column as described previously. Fractions were assayed for activation potential, combined, lyophilized, and resuspended into 100 µL of water.
Enzyme assays and cation exchange chromatography
Thirty to 150 µg of protein extract (either desalted or crude) was used for each assay. The initial assay buffer with NOS cofactors contained 1 mm NADPH, 130 µm BH4, 520 µm FMN, 200 µm FAD, 1 µm CaM, 1 mm CaCl2, 50 mm Hepes (pH 7.4), and 10 µm[14C]arginine (Amersham). Assays with desalted extracts were supplied with ADF (1–5 µL) unless indicated otherwise. Subsequently, the initial assay buffer was replaced with 50 mm NaPO4 buffer (pH 7.4) (i.e. with no NOS cofactors) and 10 µm[14C]arginine. Reactions were incubated at 30 °C for 1 h, and terminated by boiling or immediately applying the reaction to spin columns (Corning, NY, USA) containing DOWEX 50WX8-400 (Sigma) cation exchange resin. DOWEX columns were prepared as previously described , and the flow-through was counted in a scintillation counter.
Following treatment with the cation exchange resin, 10% of the unbound material was counted in a scintillation counter and the remaining 90% was used for TLC analysis as follows. The unbound material was washed with four volumes of cold acetonitrile and centrifuged for 10 min at 15 000 g (Eppendorf 5415C centrifuge; Brinkmann, Westbury, NY, USA) to precipitate large molecular weight compounds. The supernatant was evaporated to dryness in a speedvac, and resuspended in 10% of the original volume with 10% acetonitrile in water. For one-dimensional TLC, 1 µL was spotted on silica gel TLC plates (Whatman #4420221, Clifton, NJ, USA) and developed with acetonitrile/water/ammonium hydroxide 4 : 1 : 1. For two-dimensional TLC, 4 µL of this mixture was spotted on silica gel TLC plates (Whatman #4420221) and developed with n-butanol/methanol/ammonium hydroxide/water (33 : 33 : 24 : 10) in the first dimension. After drying, the plates were developed in the second dimension with chloroform/methanol/acetic acid (2 : 4 : 4). Standards of known amines and amino acids were run in parallel; they were spotted with the radioactive material and detected by spraying with ninhydrin. Radioactive arginine derivatives were detected directly on the TLC plates by autoradiography (Hyblot CL, Denville Scientific, Metuchen, NJ, USA).
We thank Dr Fujinori Hanawa for his excellent technical advice. This work was funded by grant from the National Institutes of Health (GM40672).